The primary products of fatty acid biosynthesis in most organisms are 16- and 18-carbon compounds. However, the relative ratio of chain lengths and degree of unsaturation of these fatty acids vary widely among species. Mammals and insects, for example, produce primarily saturated and monosaturated fatty acids, while most higher plants produce fatty acids with one, two, or three double bonds, the latter two comprising polyunsaturated fatty acids (PUFA's).
Two main families of PUFAs are the omega-3 fatty acids (also represented as “n-3” fatty acids), exemplified by eicosapentaenoic acid (EPA, 20:5, n-3), and the omega-6 fatty acids (also represented as “n-6” fatty acids), exemplified by arachidonic acid (ARA, 20:4, n-6). PUFAs are important components of the plasma membranes of cells, predominantly esterified in the form of phospholipids, and adipose tissue in triglycerides.
The ability of cells to modulate the degree of unsaturation in their membranes is mainly determined by the action of fatty acid desaturases. Desaturase enzymes introduce unsaturated bonds at specific positions in their fatty acyl chain substrates. Desaturase enzymes generally show considerable selectivity both for the chain length of the substrate and for the location of existing double bonds in the fatty acyl chain (Shanklin and Cahoon, 1998) and may be classified on this basis. Another classification of fatty acid desaturases is based on the moiety to which the hydrocarbon chains of their substrates are acylated. Desaturases recognize substrates that are bound either to acyl carrier protein, to coenzyme A, or to lipid molecules such as phospholipids (Murata and Wada, 1995; Shanklin and Cahoon, 1998).
The desaturation of fatty acids in glycerolipids is essential for the proper function of biological membranes. Introduction of unsaturation in the Δ9 position of palmitic or stearic acid provides fluidity to membrane lipids and thus Δ9 desaturases are found universally in living systems.
Linoleic acid (LA; 18:2, Δ9, 12) is produced from oleic acid (18:1, Δ9) by a Δ2-desaturase while α-linolenic acid (ALA; 18:3) is produced from LA acid by a Δ15-desaturase. Stearidonic acid (18:4, Δ6, 9, 12, 15) and γ-linolenic acid (18:3, Δ6, 9, 12) are produced from ALA and LA, respectively, by a Δ6-desaturase. However, mammals cannot desaturate beyond the Δ9 position and therefore cannot convert oleic acid into LA. Fourteen insect species (de Renobales, 1987) have been shown to produce linoleic acid de novo from 14C-acetate, suggesting the presence of native Δ12-desaturase activity. The house cricket Acheta domesticus Δ12-desaturase activity was the first of this type reported to utilise oleoyl-CoA (Cripps, 1990). However, no gene responsible for the conversion of oleic acid to LA has been identified from an insect despite extensive effort. Likewise, ALA cannot be synthesized by mammals. The major poly-unsaturated fatty acids of animals therefore are derived from diet via the subsequent desaturation and elongation of dietary LA and ALA. Other eukaryotes, including fungi, nematodes and plants, have enzymes which desaturate at the carbon 12 and carbon 15 positions. The membrane-associated Δ12 desaturases of Arabidopsis sp. and soybean use acyl lipid substrates
Less commonly, saturated fatty acids can be unsaturated initially at positions other than the 9-position by desaturases with unusual specificities. Petroselinic acid (cis-6-octadecenoic acid) is concentrated in the seed oils of the Umbelliferae (Apiaceae), Araliaceae and Garryaceae plant families, where it can reach 85% of the total lipid fatty acid (Kleiman and Spencer, 1982). The desaturase from coriander (Coriandrum sativum) has been characterised that makes the unusual lipid. A plastid-located acyl-acyl carrier protein (ACP) Δ4 desaturase acts on ACP-palmitic acid to produce cis-4-hexadecenoic acid which is transferred from the plastid to the developing seed where it is elongated to cis-6-octadecenoic acid (Cahoon et al. 1992). A related desaturase with 83% sequence identity has been obtained from English Ivy (Hedra helix L.) which produces cis-4-hexadecenoic acid and cis-6-octadecenoic acid when expressed in Arabidopsis (Whittle et al 2005). cis-5-Eicosenoic acid (C20:1 Δ5) is a major component of the seed oil of meadowfoam (Limnanthes alba) and related Limnathes species. The enzyme responsible for the production of the unusual oil in L. douglasii was identified as an acyl coenzyme A-Δ5 desaturase whose substrate preference is eicosanoic acid (Cahoon et al., 2000). Expression of L. douglasii Δ5 desaturase and fatty acid elongase genes in soybean embryos resulted in the production of cis-5-eicosenoic acid (C20:1 Δ5) and cis-5-docosenoic acid (C22:1Δ5). Sayanova et al (2007) have identified an acyl CoA-Δ5 desaturase which is related to the Limnathes sp. desaturase but it utilizes saturated fatty acids (C16:0, C18:0) and unsaturated fatty acids (LA, ALA) to make Δ5 monoenoic acids and polyunsaturated fatty acids. Genes encoding similar enzymes have not been cloned from animals such as insects.
Some Lepidopteran insects carry out Δ11 desaturation of saturated fatty acids (C16:0, C18:0) esterified to acylCoA as a step in the production of a diversity of moth sex pheromones (Rodriguez et al 2004). Desaturases from the moth Spodoptera littoralis were expressed in Saccharomyces cerevisiae to produce cis-Δ11 mono-unsaturated products of C14:0, C16:0 and C18:0 when the yeast cells were fed additional saturated fatty acids (Rodriguez et al 2004). In addition, trans-Δ11 tetradecenoic acid was formed from myristic acid (C14:0) fed to the yeast. A minor byproduct of the Δ11 desaturation was the formation of 11-hydroxy hexadecanoic or octadecanoic acid (up to 0.1% of total fatty acids) (Serra et al., 2006). Moto et al. (2004) identified a bi-functional acyl-CoA desaturase from the pheromone gland of the silkmoth which was responsible for the biosynthesis of the pheromone precursor. The desaturase first utilised palmitic acid to make cis-11-hexadecenoic acid and then acted on this to remove allylic 2H and form a conjugated diene fatty acid (trans-A 10,cis-Δ12-hexadecendienoic acid and some trans-Δ10,trans-Δ12-hexadecendienoic acid) and therefore it possessed both cis-Δ11 and conjugase desaturase activities. In the New Zealand leaf roller, Planotortrix octo, a desaturase has been identified from pheromone gland that desaturates palmitic acid at the Δ10 position to form cis-Δ10-hexadecenoic acid (Hao et al. 2002).
Omega-3 LC-PUFA are now widely recognized as important compounds for human and animal health and the inclusion of omega-3 LC-PUFA such as EPA and DHA in the human diet has been linked with numerous health-related benefits. These include prevention or reduction of coronary heart disease, hypertension, type-2 diabetes, renal disease, rheumatoid arthritis, ulcerative colitis, chronic obstructive pulmonary disease, various mental disorders such as schizophrenia, attention deficit hyperactive disorder and Alzheimer's disease, and aiding brain development and growth (Simopoulos, 2000). These fatty acids may be obtained from dietary sources or by conversion of linoleic (LA, omega-6) or α-linolenic (ALA, omega-3) fatty acids, both of which are regarded as essential fatty acids in the human diet. While humans and many other vertebrate animals are able to convert LA or ALA, obtained from plant sources, to LC-PUFA, they carry out this conversion at a very low rate. Moreover, most modern societies have imbalanced diets in which at least 90% of polyunsaturated fatty acid(s) consist of omega-6 fatty acids, instead of the 4:1 ratio or less for omega-6:omega-3 fatty acids that is regarded as ideal (Trautwein, 2001). The immediate dietary source of LC-PUFA such as eicosapentaenoic acid (EPA, 20:5) and docosahexaenoic acid (DHA, 22:6) for humans is mostly from fish or fish oil. Health professionals have therefore recommended the regular inclusion of fish containing significant levels of LC-PUFA into the human diet. Increasingly, fish-derived LC-PUFA oils are being incorporated into food products and in infant formula. However, due to a decline in global and national fisheries, alternative sources of these beneficial health-enhancing oils are needed.
Fatty acids may also be hydroxylated, for example ricinoleic acid (12-hydroxy-octadec-cis-9-enoic acid) which comprises up to 90% of the fatty acid in castor oil from Ricinus communis and is an important agricultural commodity oil. Other related hydroylated fatty acids found in plant oils include 12-hydroxy-octadeca-cis-9,cis-15-dienoic (densipolic) and 14-hydroxy-eicosa-cis-11,cis-17-dienoic (auricolic) acids. The Ricinus Δ12 hydroxylase acts on oleic acid lipid substrate to produce ricinoleic acid; the desaturase gene responsible for the transformation is most closely related to but divergent from plant membrane Δ12 acyl lipid desaturases (van de Loo et al 1995). There is a homologous C20 hydroxylated fatty acid produced at high levels in seed oil of Lesquerella sp. as 14-hydroxy-eicos-cis-11-enoic (lesquerolic acid) (Gunstone et al., 1994). The L. fendleri hydroxylase gene has been cloned and expressed in an Arabidopsis FAD2 mutant which accumulated ricinoleic, lesquerolic and densipolic acids in seeds (Broun et al 1998). 2-hydroxy fatty acids occur in appreciable amounts in the sphingolipids of plants and animals but they are also present as minor components of seeds oils such as 2-hydroxy-octadeca-9,12,15-trienoate from Thymus vulgaris seeds and 2-hydroxy-oleic and linoleic acids are found in Salvia nilotica (Smith, 1971; Badami and Patil, 1981).
Fatty acids may also comprise epoxy groups. The most widely known natural epoxy fatty acid is vernolic acid (12,13-epoxy-octadec-cis-9-enoic acid) from the seed oils of Vernonia spp and Euphorbia lagascae (Cuperus and Derksen, 1996). The epoxygenase gene of C. palaestina, which is related to but divergent from plant membrane Δ12-oleate desaturases, has been functionally characterized and shown to use linoleate as a substrate (Lee et al. 1998).
In some organisms, conjugated fatty acids are produced by the activity of a conjugase (Crombie et al., 1984; Crombie et al., 1985; Fritsche et al., 1999; Cahoon et al., 2001; Qiu et al., 2001). The biosynthesis of conjugated fatty acids such as calendulic acid, eleostearic acid or punicic acid proceeds via the desaturation of oleic acid to linoleic acid by a Δ12-desaturase and a further desaturation in conjunction with a rearrangement of the Z9- or Z12-double bond to the conjutrienic fatty acid by a specific conjutriene-forming desaturase (conjugase).
There is a need for further methods of producing fatty acids in recombinant cells and for more efficient production or production of novel fatty acids.
Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.